ES2392539T3 - Use of plants and plant extracts as feed additive to affect rumen fermentation - Google Patents

Abstract

Use of an additive containing one or more plant materials or plant extracts to affect rumen fermentation and to increase the availability of energy and nitrogen sources for a ruminant, characterized in that the one or more plant materials or plant extracts are selected from the group consisting of Lonicera japonica, Gentiana asclepidea, Gentiana lutea, Eugenia car and ophyllata, Bellis perennis, Olea europaea, Symphytum officinale, Carduus pycnocephalus, Paeoniae alba radix, Populus tremula, Prunus avium, Salix caprea, Rheum noum, Helum canum Arctostaphylos uva-ursi, Peltiphyllum peltatum, Epilobium montanum, Knautia arvensis, Latuca sativa and Urtica dioica, and their extracts, and! -Mircene.

Description

Use of plants and plant extracts as feed additive to affect rumen fermentation

Field of the Invention

The present invention relates to the use of an additive containing one or more components from plant materials or plant extracts in order to improve the energy or nitrogen retention of rumen fermentation, or to decrease lactic acidosis. The compounds are properly administered orally to the ruminant via animal feed or drinking water.

Background of the invention

The rumen is the first stomach of ruminants such as cattle, sheep and goats. Its structure and nutritional importance is described by Hungate (1966). The rumen allows these animals to digest plant materials with high fiber content, unsuitable for most non-ruminant animals. The rumen is a large organ, which contains an excess of 150 kg of digesta in dairy cows, in which the digestion by microorganisms is carried out. The rumen evolved in the way that it has done so that the animal benefits from the digestive activities of the microbe fiber. The final products, volatile fatty acids, are absorbed through the rumen wall, and are used for energy and protein synthesis. The main benefit of fermentation in the anterior intestine of the rumen compared to fermentation in the posterior intestine is that the microbial cells formed during fermentation pass to the curd, where they are digested and their amino acids are absorbed. Therefore, unlike fermentation in the posterior intestine, microbial amino acids are available to the host animal. Therefore, the rumen is a very efficient organ in the context of the evolution of a herbivore that subsists in poor pastures.

Fermentation of the rumen also has some disadvantages. Methane is produced as a natural consequence of anaerobic fermentation (Hungate 1966). The release of methane also represents a loss of energy from the animal. In addition, methane is a potent greenhouse gas, so ruminants damage the environment in this regard (Leng, 1992). The ruminant is also less efficient than other species in the use of a dietary protein. The nitrogen losses of ruminants are exceptionally high, particularly in grazing animals (Nolan, 1975). This is an environmental as well as an economic problem, due to the impact of nitrogen-rich droppings on the environment. In addition, there are unique digestive disorders of ruminants that occur as a direct consequence that the microbial fermentation of the rumen is altered.

Chemical additives and antibiotics for feed have been used in the past to alleviate some of these problems. The presence of such materials in the food chain is becoming less and less acceptable to regulatory authorities and to the consumer. Thus, solutions to the problems of ruminant livestock production must be natural and sustainable.

The dietary protein that enters the rumen is broken in an apparently uncontrolled manner, resulting in the formation of ammonia and subsequent loss of nitrogen in the urine (Nolan, 1975). The low efficiency of nitrogen retention, which represents a significant economic loss, causes metabolic stress in the animal, and also causes a burden on the environment, through nitrogen-rich wastes. If the rupture process could be reduced, these problems would be reduced.

In addition, the second possibility that exists to improve protein retention in the rumen is the suppression of the population of ciliated protozoa in the rumen. Protozoa consume large amounts of bacteria in the rumen, and their rupture may result in a decrease in net yield of microbial protein from fermentation in the rumen of up to 50% (Wallace et al., 1997). If protozoa could be suppressed, there would be less ammonia formation and less need for dietary protein supplementation.

Lactic acid is normally only a minor product of rumen fermentation. However, when a rapidly degraded feed is introduced too quickly, or when the concentrates form a high proportion of the diet, the production of volatile fatty acids will exceed the buffering capacity of the rumen. This can lead to a pH value so low in the rumen that only lactic acid producing bacteria can grow (Russell and Hino, 1985).

Since lactic acid is a stronger acid than volatile fatty acids, the pH drops further, the recovery of normal fermentation becomes impossible, and the animal dies. More typically, however, ruminants of highly concentrated diets suffer from subclinical acidosis, which causes suffering and decreases production efficiency although it is not life threatening. Chemical buffers can reverse this condition, but it would be better if the growth of lactic acid producing bacteria could be suppressed.

Publication WO 03/094628 describes an attempt to reduce methane production emanating from the digestive activities of ruminants by adding one or more essential oil compounds selected from the group consisting of limonene, eugenol, a salicylate, quinoline, Vanilla, thymol and a cresol. To the

At the same time, microbial activities in the rumen and the production of volatile fatty acids, for example propionic acid, could be maintained at an acceptable level.

Objects and summary of the invention

The object of the present invention is to focus generally and collectively on digestive disorders in the rumen and promote the use of available energy and nitrogen sources for ruminant growth. This object can be achieved by favoring the formation of volatile fatty acids, which are absorbed through the rumen wall and are used for energy and protein synthesis, and reducing the breakdown of dietary protein and microbial protein. Other methods are to limit methanogenesis and control or suppress lactic acid producing bacteria.

According to the invention, said object can be achieved by using an additive containing one or more components of plant materials selected from the group consisting of Lonicera japonica, Gentiana asclepidea, Gentiana lutea, Eugenia caryophyllata, Bellis perennis, Olea europaea, Symphytum officinale , Carduus pycnocephalus, Paeoniae alba radix, Populus tremula, Prunus avium, Salix caprea, Rheum nobile, Helianthemum canum, Arctostaphylos uvaursi, Peltiphyllum peltatum, Epilobium montanum, Knautia arvensis, Latuca sativa and Urtica dioica, and their extracts. The total amount of the components to be administered to an animal is suitably from 0.02 mg to 20 g per day and kg of body weight.

It has been shown that all components contribute essentially to the efficiency of fermentation in the rumen without having any considerable inconvenience. Some of the components suppress the activity of protozoa, and others decrease proteolytic activity, methanogenic activity or lactic acid production.

Detailed description of the invention

Since the components influence fermentation in different ways, it is advantageous to combine specific compounds to optimize fermentation according to the object of the invention.

Extensive studies have shown that the components of a subgroup consisting of plant materials of Helianthemum canum, Arctostaphylos uva-ursi, Epilobium montanum, Knautia arvensis and Peltiphyllum peltatum, and their extracts, reduce rumen proteolysis. In addition, all these components inhibit the activity of protozoa, and most of them also have beneficial influences on other essential parameters of fermentation, such as methane production, the amount of volatile fatty acids, digestibility, microbial biomass and / or fermentation efficiency. The most suitable components are plant materials from Arctostaphylos uva-ursi, Knautia arvensis and Helianthemum canum, and their extracts. The most preferred components are Knautia arvensis plant materials and their extracts, especially methanolic extracts.

Plant materials from Lonicera japonica, Gentiana asclepidea, Gentiana lutea, Eugenia caryophyllata, Bellis perennis, Olea europaea and Symphytum officinale, and their extracts, and! -Mircene, form another subgroup, whose components suppress the bacteriolytic activity of ciliated protozoa in the rumen fluid Eugenia caryophyllata also increases microbial biomass and reduces methane production. Bellis perennis has a small reduction in methane production, and also improves the efficiency of fermentation and the production of microbial biomass, while Symphytum officinale increases the efficiency of fermentation and reduces the effects of acidosis. ! -Mircene has a moderate inhibitory effect on protozoic activity, but also finds use for the reduction of methane production. An extract prepared from the flowers of Lonicera japonica eliminated the bacteriolytic activity of ciliated protozoa. A seed preparation of Lonicera japonica also reduced protozoic activity, but only up to about 60%. Preferred components of this subgroup are plant materials of Bellis perennis, Eugenia caryophyllata, Olea europaea and Symphytum officinale, and their extracts, especially the methanolic and aqueous extracts of Bellis perennis and Gentiana asclepidea.

The plant materials of Carduus pycnocephalus, Paeoniae alba radix, Populus tremula, Prunus avium, Salix caprea, and Rheum nobile, and their extracts, form a third subgroup, whose components are used appropriately to decrease methanogenic activity in the rumen digesta . For these components no significant detrimental effects on other fermentation parameters have been observed. Preferred components are the plant materials of

Carduus pycnocephalus, Populus tremula, and Rheum nobile, and their extracts, Carduus pycnocephalus and Rheum nobile are especially preferred.

Finally, a fourth subgroup consists of plant materials of Latuca sativa and Urtica dioica, and their extracts. These components reduce the activity and / or the formation of lactic acid. Latuca sativa, sometimes called Latuca virosa, also decreases methane production and protozoic activities, and increases the formation of volatile fatty acids. Dioctic urtica also has additional effects, and inhibits proteolysis and protozoic activities, and promotes the formation of volatile fatty acids.

In a suitable embodiment of the invention, the additive contains components of more than one of the subgroups defined above, and it may be favorable for the additive to contain components of the four subgroups. Advantageously, the additive contains at least one component selected from each of the groups, which consists of

i) plant materials of Knautia arvensis, Arctostaphylos uva-ursi and Helianthemum canum, and their extracts,

ii) plant materials of Bellis perennis, Eugenia caryophyllata, Olea europaea, Lonicera japonica Symphytum officinale, and their extracts,

iii) plant materials of Carduus pycnocephalus, Populus tremula, and Rheum nobile, and their extracts, and

iv) plant materials of Latuca sativa and Urtica dioica, and their extracts. The total amount of the components belonging to the groups i) -iv) is suitably 20-100% by weight, preferably 40100% by weight of the total amount of the components present according to the invention.

Extracts of plant materials according to the invention include, but are not limited to, concretes, essential oils, resinoids, tinctures, absolutes, and absolute oils. When plant extracts or plant parts are produced, water, organic solvents, and mixtures thereof can be used, according to conventional methods, but dry distillation can also be applied. Examples of suitable organic solvents are methanol, ethanol, propanol, butanol, ethyl acetate, methyl ethyl ether, diethyl ethers, methylene chloride, chloroform, carbon tetrachloride, benzene, toluene, petroleum ether and acetone.

The total amount of the components administered to the animal depends on whether the components are an essential oil compound, extracts of plant material, or plant material. In the case that the components are all myrcene, and / or extracts, then the amount administered is normally 0.1-50 mg per day and kg of body weight, whereas, when the components consist only of plant material, The amount administered is usually 0.02-10 g per day and kg of body weight.

The additive may contain ingredients other than the components of the invention. Suitably, the additive contains 0.1-100%, preferably 0.2-90% by weight of the components, as well as agents that enhance growth, flavorings, absorbent supports and / or other feed ingredients. Preferably, the total amount of growth enhancing agents, flavorings, absorbent supports and / or other feed ingredients is 10 to 75% by weight of the additive. In the case where any of the components is plant material, they are properly dried and ground to a particulate material or powder before they are mixed with the other ingredients in the additive, if any.

Examples of suitable growth-enhancing additives and flavorings are cresol, anethole, deca-, undecay / or dodecalactones, ionones, irone, gingerol, piperidine, propylidenephthalide, butylidenephthalide, capsaicin and / or tannin. The support may contain, for example, 40-50% by weight of wood fibers, 8-10% by weight of stearin, 4-5% by weight of turmeric powder, 4-5% by weight of rosemary powder, 22 -28% by weight of limestone, 1-3% by weight of a gum, such as gum arabic, 5-50% by weight of sugar and / or starch, and 5-15% by weight of water. The other feed ingredients are properly selected from the group consisting of vitamins, enzymes, mineral salts, ground cereals, protein-containing components, components that contain carbohydrates, molds and / or wheat bran. The additive is suitably added to the feed composition according to the invention in amounts such that the feed will normally contain 0.4 ppm-80% by weight of the components.

The feed composition usually contains, calculated on the dry weight of the feed, the following ingredients:

a) 0-80% dry weight of cereals,

b) 0-30% dry weight of fat,

c) 0-85% by dry weight of nutritional substances containing protein of a different type of cereals, and

d) 10 ppm-40% dry weight of the components of the invention.

The total amounts of a) -d) are preferably at least 50% dry weight.

When the feed composition is prepared, the additive can be mixed with dry ingredients consisting of cereals, such as ground or crushed wheat, oats, barley, corn and rice; vegetable protein sources based on, for example, rapeseed, soy bean and sunflower seed; sources of animal protein; molasas; and dairy products, such as various milk powders and whey powders. After mixing all the dry ingredients, you can add the liquid ingredients and the ingredients, which after heating make them liquid. The liquid ingredients may consist of lipids, such as fat, for example vegetable fat, optionally liquefied by heating, and / or may consist of carboxylic acids, such as a fatty acid. After thorough mixing, a mealy or particulate consistency is obtained, depending on the degree of grinding of the ingredients. To avoid separation during use, water should preferably be added to animal feed, which is then subjected to a conventional pelletizing, expansion or extrusion process.

Any excess water can be removed by drying. If desired, the resulting granular animal feed can also be crushed to a smaller particle size. The described feed composition is usually administered in combination with dry green fodder and / or silage.

The drinking water supplement may contain at least 1%, suitably 1-99% by dry weight,

5 preferably 10-50% dry weight of the components. In addition to one or more of the components, the supplement may also contain 1-99% dry weight of a large number of other ingredients. Suitable examples of other ingredients are mineral salts, vitamins, additives that improve health and growth, flavorings, water soluble or water dispersible carriers, such as sugars, milk powder, milk by-products and cellulose derivatives, dispersing agents and stabilizers, such as soluble polymers

10 in water or water dispersible, and mixtures thereof. When the drinking water is prepared, the supplement is normally added to the water in an amount such that the concentration of the components is 1 ppm-10% by weight.

Within the scope of the invention, it is also possible to produce a suspension of the feed composition. This is especially convenient if the feed is prepared for immediate consumption.

The present invention will now be further illustrated by the following Examples.

15 Examples

In the present Examples, a number of tests were carried out with samples of plant materials, extracts of plant materials, and an essential oil compound. Samples of plant materials were lyophilized and ground, and passed through a 1 mm sieve. The samples were as follows.

Sample No.

Botanical name Sample Description

one

Arctostaphylos uva-ursi Leaves and stem

2

Bellis perennis Plant, mainly leaves

3

Carduus pycnocephalus Mix of stems, leaves and flowers

4

Epilobium montanum Foliage

5

Eugenia caryophyllata Dry embryonic seed

6

Gentiana asclepidea Leaf and stem preparation

7

Helianthemum canum Leaves and flowers

8

Knautia arvensis Scabious, completely on the ground

9

Latuca sativa Garden lettuce, all on the ground

10

Lonicera japonica Leaves, stems and flowers

eleven

Lonicera japonica (flower) Flower extract

12

Lonicerajaponica + Magnolia officinalis Dried leaves and flowers

13

! -Myrcene Essential oil compound

14

Olea europaea Dry leaves

fifteen

Paeoniae alba radix Root

16

Peltiphyllum peltatum Completely on the ground

17

Populus tremula Leaf and stem preparation

18

Prunus avium Mainly leaves and small stems

19

Rheum nobile Leaves and stem

twenty

Salix caprea Mainly leaves and small stems

twenty-one

Symphytum officinale Plant completely on the ground

22

Dioecious urtica Nettles

The effects on the rumen digesta of samples 1-22 according to the present invention were tested with respect to the bacteriological activity of ciliated rumen protozoa, the proteolytic activity of the rumen digesta, the formation of methane caused by digestion from rumen, to lactic acidosis and the formation of volatile fatty acids, as well as with respect to other effects on fermentation. The following methodologies were applied in the trials.

Bacteriolytic activity of rumen ciliated protozoa

The rupture rate of Selenomonas ruminantium labeled with [14C] leucine was determined by incubating in vitro ruminal fluid cast with labeled S. ruminantium in the presence of 5 mM unlabeled L-leucine, as previously described (Wallace and McPherson, 1987). Under these conditions, the vast majority of bacterial protein breakdown is caused by ingestion and digestion of bacteria by ciliated protozoa. Except as otherwise noted, samples were added at a concentration of 5 mg / ml. The sheep received a mixed diet of grass hay: concentrate (Frumholtz et al., 1989).

Persistence of antiprotozoic activity

The samples were pre-incubated with Coleman buffer, rumen fluid or water for 24 h, and then subsequently incubated with recent ruminal fluid in the bacteriolysis assay described above.

Proteolytic activity of the rumen digesta

Method 1. To determine the proteolytic activity using the Wallace 14C-casein method (1983), cast ruminal fluid from the same sheep was used. Samples were added to a concentration of 2.5 mg / ml, and an incubation time of 1 h was used.

Method 2. Based on Hoffman et al. (2003a, b). As donor animals, five fistulated lactating Holstein cows, fed on a typical dairy cow diet, were used. Fluid was manually extracted from the rumen before morning feeding. The rumen fluid was filtered through a 100 µm nylon mesh and mixed with 10% incubation buffer (v / v). The rumen buffered fluid in a volume of 75 ml was dispensed in the incubation bottles (RPT) containing a substrate mixture of 450 mg of corn silage, 225 mg of barley grain (both ground to pass a sieve of 1 mm), and a protein supplement consisting of 150 mg of fat-free crude soy bean flour and 10 mg of bovine serum albumin. This substrate, without the addition of the test plants, was incubated as a negative control. When the test plants were added, they replaced the silage. The monensin used as a positive control was dissolved in ethanol (14 mg / ml), and 11.25 µl of this stock solution was dosed in each flask immediately after filling them to a final concentration of 3 µM monensin. All flasks were incubated at 39 ° C for 12 h. Two replicates of each treatment were taken as samples at regular intervals, while a third sub-replica was reserved for gas reading. At each sampling time, 2 x 900 µl of the incubation bottle was extracted with vigorous stirring and prepared for the determination of the concentration of SCFA, ammonia and protein. SDS-PAGE was carried out by the method of Laemmli, and the quantification of total protein by point transfer according to Hoffman et al. (2002).

General effects on ruminal fermentation

Hohenheim methods for general effects on fermentation

Experimental design:

Incubation system: Reading Pressure Technique (RPT), 75 ml, total experiment time 24 h

Donor animals: 3 non-lactating Holstein cows

Basal Substrate: Grass Silage 0.750 g (control)

Inclusion level of test plants: 10% (silage replacement)

Gas readings: 2, 4, 6, 8, 10, 12, 16, 24 h

Sampling: 24 h for SCFA and TD (NDS treatment in nylon bags)

6, 8, 10 h for SCFA (900 μl) and RNA (30 μl, maximum analyzed).

Parallel: 3 independent experiments (3 different donor animals) in duplicates

RPT buffer Components Molarity Final conc. Ammonium bicarbonate 60.1 13.5 mM Sodium bicarbonate 84.0 86.5 mM Disodium hydrogen phosphate 142.0 5.5 mM Potassium dihydrogen phosphate 136.1 9.5 mM Magnesium sulfate 7xH2O 246.5 0.5 mM Microminerals 0.020% Resazurine 1% 0.001% d H2O Reducing solution 6% Total volume of rumen fluid 10%

Reducing solution

MW Components Final Conc.

Cysteine HCI 52.9 0.118 M

1M NaOH 40.0 0.040%

Na2S 240.2 0.026 N

Total volume complete with dH2O

0.5x Microminerals

MW Components Final Conc.

Calcium Chloride 2xH2O 147.0 0.45 M

Manganese Chloride 4xH2O 197.9 0.25 M

Cobalt Chloride 6xH2O 237.9 0.02 M

Ferric Trichloride 6xH2O 270.3 0.15 M

Total volume complete with dH2O

Detailed description of the methods:

In vitro incubation

The rumen fluid was collected from cannulated cows that received a mixture of grass silage hay at a

5 maintenance feed in two portions equal to 8:00 and 16:00. The rumen fluid was collected before feeding in thermos bottles, filtered through a 100 µm nylon mesh and added to a mineral solution with reduced buffer. All stages were carried out at 39 ° C in CO2, to maintain anaerobic conditions.

The substrates were incubated at a ratio of 10 mg / ml of medium, that is, 750 mg of substrate was incubated with 75 10 ml of buffered rumen fluid containing 7.5 ml of filtered rumen fluid (10% v / v of rumen fluid).

Gas volume or pressure was frequently recorded at 2, 4, 6, 8, 10, 12, 16, and 24 hours of incubation, and samples were taken after 6, 8, 10, and 24 hours.

Analysis

The gas pressure was recorded in the serum bottles of the RPT system and converted to volume using an experimentally determined calibration curve. After each measurement, the gas was released from the serum bottles. True in vitro digestibility was determined by transferring the incubation content to previously weighed polyester bags (Ankom 51 µm pore size). The bags were heat sealed, cooked in neutral detergent solution for 1 hour, dried at 105 overnight, and weighed for digestibility determination. SCFAs were determined by gas chromatography using a stainless steel column packaged with GP 10% SP 1000 1% H3PO4, Chromosob W AW (Suppelco Inc. Bellafonte, PA). To 0.9 ml of the incubation supernatant was added 0.1 ml of formic acid containing the internal standard (1% methylbutyric acid). The proteins were precipitated overnight at 4 ° C. The samples were centrifuged

(30,000 g, 10 min., 4 ° C), and the supernatant was collected for analysis in appropriate GC vials.

The RNA was extracted according to the modified phenol-chloroform extraction protocol. 600 µl of phenol pH 5.1, 270 µl of pH 5.1 buffer, 30 µl of SDS (20% w / v) and 1.0 g of beads were added to the samples (300 µl) Zirconium The cells were lysed by hitting the samples for 2 x 2 minutes in a pearl mixer (50 Hz), interrupted by an incubation for 10 min. at 60 ° C. The samples were cooled on ice for 10 min., And 300 µl of chloroform was added. After stirring vigorously and another 10 min. at RT, the samples were centrifuged (10,000 g, 5 min., 4 ° C) to separate the aqueous and organic phases. The aqueous phase was quantitatively removed and transferred to a vial containing 300 µl of ammonium acetate (7.5 M) and 900 µl of isopropanol. Samples were incubated overnight at -20 ° C, and nucleic acids were precipitated by centrifugation.

(16,000 g, 10 min., 4 ° C). The supernatant was discarded, and the samples were washed once in 80% ethanol. Nucleic acids were loaded on agarose gels and densitometrically quantified after staining with ethidium bromide against a calibration curve (25-300 ng / )l).

Reading methods for general effects on fermentation

A series of thirteen consecutive fermentations, one per week, was carried out using the Reading Pressure Technique [RPT], to identify the potential effects of the inclusion of these test materials on the fermentation and degradation characteristics of a basal forage [corn silage]. All materials were examined in triplicate and at two levels of inclusion [100 and 400 mg g-1 of DM substrate], with the exception of! Myrcene [inclusion levels of 10 and 40 mg g-1]. The corn silage was previously dried and ground to pass a 2 mm sieve. A total of 1.0 g of mixed substrate [silage plus substrate] was placed in each fermentation flask. Buffered incubation medium [90 ml] was then added, the flasks sealed and stored overnight at room temperature. Before inoculation with prepared rumen fluid, the flask contents were heated to 39 ° C. The rumen fluid was obtained manually [hand-squeezed contents] before feeding [07.00] of two lactating dairy cows that were offered at will a ration of corn / grass silage: concentrated [60:40]. The fluid was filtered through two layers of muslin, and kept in CO2 at 39 ° C until use. To each flask were added 10 ml of prepared fluid, and incubated at 39 ° C throughout the study. In each experiment, six negative controls [buffer medium plus rumen fluid only] were included to correct the gas values and the degradation residues for the direct effects of the rumen fluid. In addition, in all experiments [six flasks per experiment], an unsupported corn silage [1.0 g flask-1] was included. The results in the studies were expressed with respect to the positive control values.

Gas pressure readings from the upper space were obtained 2, 4, 6, 8, 10, 12, 14, 16, 18, 21 and 24 hours after inoculation. After the last measurement, approximately 3.0 ml of fermentation medium from each flask was taken as a sample and volume was increased by test material / inclusion level and stored frozen [-20 ° C] until further analysis to determine the VFA composition using a Varian 3600 GC gas chromatograph. Fermentation residues were recovered by filtering the contents of the flask through 60 ml Gooch crucibles [porosity 1, 100 to 160 µm] in a light vacuum. The residues were then dried [100 ° C for 24 hours], weighed, pyrolyzed [500 ° C overnight] and weighed again. The amount of dry matter [DM] and organic matter [OM] determined was used to evaluate iDMD and iOMD [in vitro degradation of DM and OM, respectively]. The degree [ml of g-1 OM gas incubated] and the rate of fermentation gas release [ml h-1] were generated using a quadratic function previously derived to convert the pressure to volume. The fermentation efficiency [FE] of the feed was estimated as iDMD [g kg-1] / accumulated gas release [ml] at 24 h after inoculation. Statistical differences between treatment means and positive control [corn silage alone] were evaluated using the Student t test and the significance declared at P> 0.05.

Lion methods for general effects on fermentation

Sheep with cannulated rumen, fed a diet consisting of 75% alfalfa hay and 25% barley (free access to vitamin and mineral supplements and water), were used as donors for rumen fluid. The rumen fluid was collected just before the morning meal.

The substrate used for batch crops consisted of 50% alfalfa hay, 40% grass hay and 10% barley grain, ground in a hammer mill with a 1 mm sieve. The amount of substrate used was 500 mg, and the inclusion rate of the test plant was 10% (approx. 50 mg). Both were weighed in serum bottles, in which 50 ml of buffered ruminal fluid (containing 10 ml of ruminal fluid were anaerobically dispensed)

filtered and 40 ml of the medium described by Goering and Van Soest, 1970). The bottles were sealed and incubated at 39 ° C. After 24 h of incubation, the following measures were recorded:

-

Total gas production (ml), using a pressure transducer and collecting the gas in a calibrated syringe.

-

pH in the incubation medium.

-

Concentration of volatile fatty acids in the incubation medium, by GC (the VFA was also measured at the inoculation time to calculate the production of VFA after 24 h).

-

DM incubation residue, filtering the contents of the bottles in sintered crucibles and weighing the

residue after drying. Then, the neutral detergent fiber (NDF) content of the residue was determined

to calculate the amount of NDF without degrading. From this, the disappearance of DM and NDF was estimated.

Three replicates were incubated per plant, and blanks (without substrate or plant) and controls (500 mg of substrate without any plant, and 550 mg of substrate without any plant) were used.

Methane formation by rumen digesta

Incubations carried out by the general method of León were analyzed to determine methane formation. A representative sample of the gas produced was taken for subsequent analysis. Sampling was carried out using special gas-tight glass syringes, equipped with a valve that allows the sample to be enclosed and stored. The sample was collected directly from the upper space, after inserting the needle through the septum of the cap, taking a sample in the syringe and closing the valve. This sample was collected after measuring the total gas production, assuming that the gas consumption was the same in the gas released and in the one remaining in the upper space.

The methane concentration in the gas produced was determined by gas chromatography. The gas sample (300 to 500 µl) was injected directly from the gas-tight syringe to the corresponding port. The configurations of the GC parameters used for the analysis were:

Instrument: Shimadzu GC-14B provided with a flame ionization detector (FID)

Column: stainless steel column 2.3 meters long x 2.1 mm internal diameter packed with stationary phase of Carboxen 1000 mesh 60/80

Carrier gas: helium, flow rate (100 kPa), constant flow mode

Temperatures:

Detector: FID. Temperature 200ºC, synthetic air flow (50 kPa), H2 flow (50 kPa)

Injector: Temperature 200ºC

Column oven: 170 ° C (isothermal)

The standard used for calibration was pure methane (99.9%). The standard curve was established by graphically representing a linear regression of the injected amounts of methane against the area of the peaks to obtain the response factor.

Acidosis Assay

A model was used in which the decrease in the pH of the incubation medium and the concentration of lactic acid produced, with respect to the controls during a 48 h fermentation period, provided an indication of the ability of the test substrates to counteract the potential acidic effects. In this study, ground wheat grain [2 mm] was used as the basal substrate, and the test materials were included at 100 mg g-1 DM [or 10 mg g-1 DM! -Mircene]. As before, 1.0 g of total substrate was added to each flask. To improve the production of lactic acid, the prepared buffer solution was diluted to 50 percent. This was done to ensure that the buffering capacity of the incubation medium was exceeded, and that the resulting decrease in pH encouraged the growth of lactobacilli and other lactic acid producing bacteria. Ruminal fluid was obtained and prepared as previously described, except that the two cows used were offered an early lactation ration with an elevated level of coarse ground wheat. Fluid pH measurements were taken one hour after inoculation [starting pH], and after 23 and 47 hours later, inserting a pH electrode directly into each flask. Immediately after the last measurement, approximately 3.0 ml of fluid was taken from each flask, increased in volume by the sample and frozen stored at -20 ° C until it was enzymatically analyzed for the determination of L [+] lactic acid.

The results are presented as absolute differences with respect to the controls. A positive effect on acidosis was identified as a decrease in the reduced pH rate at 24 h, a higher endpoint pH and a reduced concentration of lactic acid.

Results

5 The results obtained from the measurements of the fermentation activities are shown in Tables 2a, 2b and 2c below.

The results obtained in the protozoan test, the proteolytic test by method 1, and in the formation of methane, are shown in the same tables.

The effects of selected plants / plant extracts at different inclusion rates on the activity

10 bacteriolytic protozoa in vitro are shown in Fig. 1 to Fig. 7. The persistence of the bacteriolytic activity of the protozoa was greater in ruminal fluid than in buffer or in water, particularly with G. asclepiadea (Table 1) . The influence of K. arvensis is shown in Fig. 8.

Table 1. Influence of pre-incubation in rumen fluid or Coleman buffer on the ability of samples to inhibit bacteriolytic activity of rumen ciliated protozoa

Bacteriolytic activity (% activity in control without addition or Bellis perennis Gentiana asclepiadea pre-incubation)

Control, without addition 100 100

+

additive, without preincubation 0 42

+

 additive, 24h preincubation in RF 0 20

+

additive, 24h preincubation in Coleman buffer 13 28

+

 additive, 24h preincubation in water 24 68

Table 2a. Effects of plant materials, plant extracts or components of identical nature selected on fermentation. The missing values of plant material are 100.

General effects on fermentation

Sample No.

Amount in feed,%

one 2 3 4 5 6 7 8

Reading Diet

Dry matter digestion

10 95 97 99 93 102 85 99

40

82 106 99 84 98 43 96

Cumulative gas, 24 h

10 86 97 92 85 96 79 96

40

72 88 88 68 92 42 90

Fermentation efficiency

10 110 100 108 110 106 107 103

40

114 121 112 124 106 101 107

Hohenheim Diet

Digestibility

10 102 102 103 103 103 101 103 102

Gas production

10 105 102 108 108 103 104 108 99

Fermentation efficiency

10 97 100 95 95 99 97 95 102

SCFA

10 110 118 124 124 106 111 119 96

General effects on fermentation

Sample No.

Amount in feed,%

one 2 3 4 5 6 7 8

C3 / C2

10 106 102 108 108 95 97 107 96

Leon's diet

Digestibility

10 104 105 106 106 97 102 103 105

Digestibility ap.

10 95 98 97-107 101 94 98 96 101

Gas production

10 105 103 104 104 102 104 103 96

Fermentation efficiency

10 100 102 102 102 96 98 101 115

Microbial biomass

10 104 108 110 110 98 97 105 125

C3 / C2

10 101 100 95 95 102 100 99 96

TV FA

10 108 102 106 106 90 97 104 108

Specific effects on fermentation

Protozoic activity

5 mg / ml 87 7 84 70 2 4 73 82

Methane Formation, Leon

10% feed 83 94 70-92 102 93 106 90 97

Proteolysis, Method 1

79 90 87 76 102 104 78 105

Heartburn, 24h 48h

99 77 76 88 61 79 80 89

100

77 71  100 51 82 70 84

Table 2b Effects of plant materials, plant extracts or components of identical nature selected on fermentation. The missing values of plant material are 100 Table 2c. Effects of plant materials, plant extracts or components of identical nature selected on fermentation. The missing values of plant material are 100

General effects on fermentation

Sample No.

Amount in feed,%

9 10 eleven 12 13 14 fifteen 16

Reading Diet

Dry matter digestion

10 102 99 98 97 103 92 104 90

40

109 106 98 89 89 89 112 49

Cumulative gas, 24 h

10 95 95 91 90 100 84 100 88

40

79 91 82 82 82 63 107 51

Fermentation efficiency

10 107 104 108 107 103 110 104 102

40

139 116 119 109 109 141 104 96

Hohenheim Diet

Digestibility

10 102 100 97 99 100 101 99 102

Gas production

10 100 98 99 106 101 111 104 103

General effects on fermentation

Sample No.

Amount in feed,%

9 10 eleven 12 13 14 fifteen 16

Fermentation efficiency

10 102 102 98 94 99 91 94 99

SCFA

10 98 105 97 103 103 110 105 118

C3 / C2

10 98 98 97 98 104 103 100 102

Leon's diet

Digestibility

10 102 105 95 97 102 100 104 103

Digestibility ap.

10 96 100 98 97 93 93 110-113 107

Gas production

10 107 98 98 99 97 106 95 98

Fermentation efficiency

10 97 106 98 99 109 96 117 105

Microbial biomass

10 102 112 93 94 115 95 116 110

C3 / C2

10 105 111 106 105 105 109 105 95

TVFA

10 105 94 99 103 96 108 120 97

Specific effects on fermentation

Protozoic activity

5 mg / ml 67 40 18 5 68 12 103 83

Methane Formation, Leon

10% feed 86 108 106 108 90 100 46-92 93

Proteolysis, Method 1

154 112 113 92 110 70 83 73

Heartburn, 24h 48h

58 106 91 87 105 97 108 66

63

100 92 82 115 91 109 91

General effects on fermentation

Sample No.

Quantity, %

17 18 19 twenty twenty-one 22

Reading Diet

Dry matter digestion

10 96 99 97 97 93 99

40

97 98 90 81 90 89

Cumulative gas, 24 h

10 93 98 90 89 87 90

40

84 89 64 76 80 78

Fermentation efficiency

10 104 100 107 109 108 110

40

115 110 141 107 112 115

Hohenheim Diet

Digestibility

10 100 101 97 102 101 100

General effects on fermentation

Sample No.

Quantity, %

17 18 19 twenty twenty-one 22

Gas production

10 99 103 96 102 105 99

Fermentation efficiency

10 101 98 102 101 96 101

SCFA

10 107 101 98 103 105 107

C3 / C2

10 100 93 100 91 97 100

Leon's diet

Digestibility

10 99 103 102 105 99 99

Digestibility ap.

10 97-105 90-106 89-106 78-102 78 95

Gas production

10 95 100 104 109 98 95

Fermentation efficiency

10 104 104 99 96 100 104

Microbial biomass

10 102 107 107 102 96 102

C3 / C2

10 103 92 107 99 86 103

TVFA

10 104 105 98 103 93 104

Specific effects on fermentation

Protozoic activity

5 mg / ml 84 83 57 76 13 83

Methane Formation, Leon

10% feed 74-89 79-85 75-78 70-86 78 93

Proteolysis, Method 1

2.5 mg / ml 87 120 97 97 105 83

Heartburn, 24h 48h

78 82 69 72 14 77

73

74 53 56 58 79

From the results obtained, it is evident that the tested components of the invention have positive effects on rumen fermentation, effects which will support the objects of the present invention.

Cited bibliography

5 Hoffman EM, Meutzel S, Becker K (2002), A modified dot-blot method of protein determination applied in the tanninprotein-precipitation assay to facilitate the evaluation of tannin activity in animal feeds, Br. J. Nutr. 87: 421-426

Hoffman EM, Meutzel S, Becker K (2003), Effects of Moringa oleifera seed extract on rumen fermentation in vitro, Arch. Anim. Nutr. 57: 65-81

Hoffman EM, Meutzel S, Becker K (2003), The fermentation of soybean meal by rumen microbes in vitro reveals 10 different kinetic features for the inactivation and the degradation of trypsin inhibitor protein, Anim. Feed Sci. Technol.

106: 189-197

Hungate RE (1966), The rumen and its microbes, Academic Press, New York and London Leng RA (1992), Ruminant production and greenhouse gas emissions, Proc. NZ Soc. Anim. Prod. 52 (Suppl.): 15-23

Nagaraja TG, Newbold CJ, Van Nevel CJ, Demeyer Dl (1997), Manipulation of ruminal fermentation, in The rumen 15 microbial ecosystem, ed. Hobson PN and Stewart CS, Chapman & Hall, London p. 523-632

Nolan JV (1975), Quantitative models of nitrogen metabolism in sheep, in Digestion and Metabolism in the Ruminant, ed. McDonald IW and Warner ACI, University of New England Publishing Unit, Armidale, Australia p. 416-431

Russell JB, HinoT (1985), Regulation of lactate production in Streptococcus bovis: aspiraling effect that contributes to rumen acidosis, J. Dairy Sci. 68: 1712-1721

Wallace RJ, McPherson CA (1987), Factors affecting the rate of breakdown of bacterial protein in rumen fluid, Br. J. Nutr. 58: 313-323

Wallace RJ (1983), Hydrolysis of 14 C-labeled proteins by rumen micro-organisms and by proteolytic enzymes prepared from rumen bacteria, Br. J. Nutr. 50: 345-355

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Claims (10)

one.

 Use of an additive containing one or more plant materials or plant extracts to affect rumen fermentation and to increase the availability of energy and nitrogen sources for a ruminant, characterized in that the one or more plant materials or plant extracts are selected from the group consisting of Lonicera japonica, Gentiana asclepidea, Gentiana lutea, Eugenia caryophyllata, Bellis perennis, Olea europaea, Symphytum officinale, Carduus pycnocephalus, Paeoniae alba radix, Populus tremula, Prunus avium, Salix caprea, Rheum noumva, Heliantumumumumumumum, Heliantum -ursi, Peltiphyllum peltatum, Epilobium montanum, Knautia arvensis, Latuca sativa and Urtica dioica, and their extracts, and! -mirceno.

2.

 Use according to claim 1, characterized in that the selected components contain one or more components of the subgroup consisting of plant material of Helianthemum canum, Arctostaphylos uva-ursi, Epilobium montanum, Knautia arvensis and Peltiphyllum peltatum, and their extracts, to decrease proteolytic activity of ruminal digestion.

3.

Use according to claim 1 or 2, characterized in that the selected components contain one or more components of the subgroup comprising plant materials of Lonicera japonica, Gentiana asclepidea, Eugenia caryophyllata, Bellis perennis, Olea europaea and Symphytum officinale, and their extracts, and! - Myrcene, to suppress the activity of protozoa.

Four.

 Use according to any one of claims 1-3, characterized in that the selected components contain one or more components of the subgroups consisting of plant materials of Carduus pycnocephalus, Paeoniae alba radix, Populus tremula, Prunus avium, Salix caprea, and Rheum nobile, and its extracts, to decrease the methanogenic activity of rumen digestion.

5.

 Use according to any one of claims 1-4, characterized in that the selected components contain one or two of the subgroup components consisting of plant materials of Latuca sativa and Urtica dioica, and their extracts, to decrease the production of lactic acid.

6.

Use according to claims 2-5, characterized in that the additive contains components of at least two of the subgroups defined in claims 2-5.

7.

Use according to claim 6, characterized in that it contains components of all subgroups defined in claims 2-5.

8.

 Use according to claim 7, characterized in that at least one of the components is selected from each of the groups i) consisting of plant materials of Knautia arvensis, Arctostaphylos uva-ursi and Helianthemum canum, and their extracts, ii) consisting of plant materials of Bellis perennis, Eugenia caryophyllata, Olea europaea, Lonicera japonica Symphytum officinale, and their extracts, iii) consisting of plant materials of Carduus pycnocephalus, Populus tremula, and Rheum nobile, and their extracts, and iv) consisting of materials Latuca sativa and Urtica dioica vegetables, and their extracts.

9.

Use according to claim 8, characterized in that the total amount of the components belonging to the groups i) -iv) is suitably 20-100% by weight of the total amount of the components present in the additive.

10.

Use according to claims 1-7, characterized in that the total amount of the components to be administered is 0.02 mg to 20 g per day and kg of ruminant body weight.

Fig. 1. The effects of Gentiana asclepiadea at different inclusion rates (/g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 2. The effects of Lonicera japonica at different inclusion rates (∀g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 3. The effects of Eugenia caryophyllata at different inclusion rates (/g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 4. The effects of Bellis perennis at different inclusion rates (/g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 5. The effects of European Olea at different inclusion rates (∀g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 6. The effects of Symphytum officinale at different inclusion rates (/g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 7. The effects of! -Mircene at different inclusion rates (∀g / ml) on the bacteriolytic activity of ciliated protozoa of the rumen in vitro. Fig. 8: The quantification of protein degradation by point transfer test confirms the inhibition of proteolysis by two different K. arvensis inputs.